It has long been known that men and women with significant antibody titers against human sperm are often infertile or have reduced fertility without other ill effects (Ingerslev and Ingerslev, 1989; Chen and Jones, 1981; Menge et al., 1982; Bronson et al., 1984). It has also been demonstrated that immunization of male and female animals with extracts of whole sperm can induce infertility (Kummerfeld and Foote, 1976; Munoz and Metz, 1978; Tung et al., 1979; Menge et al., 1979). Primakoff et al. (1988a) used a monoclonal antibody to purify a sperm-specific guinea pig surface antigen, PHG-20, and were able to demonstrate that injection of this purified antigen into either male or female guinea pigs induced a long-lasting immunity against fertilization (1988b). In previous studies it was shown that this monoclonal antibody reacted with a sperm adhesin to block its interaction with the zona pellucida of the egg, an essential step for fertilization (Primakoff et al., 1985).
A number of other monoclonal antibodies, prepared to ejaculated human sperm, or prepared to sperm of other species, have been found to cross react with human sperm. Some react with components of seminal plasma and others recognize antigens of testicular origin. Monoclonal antibodies which can immobilize or agglutinate human sperm or inhibit sperm binding and penetration of zona-free hamster ova have been reported. At present, several human sperm antigens are known such as the M.sub.r 95,000 antigen of Moore (Moore et al., 1987); the 55 kDa antigen recognized by the S36-37 mAbs (HSA-63) of Lee (Liu et al., 1990); and human homologs of 95 kDa and 56 kDa sperm receptors for ZP-3 (defined in mice by Saling and Bliel and Wassarman (Bliel, 1990; Leyton and Saling, 1989)). Also of interest is the FA-1 antigen of mouse and humans, partially characterized by Naz (Naz, 1988) and the 24 kD antigen from rat and human testis characterized by Shaha (Shaha et al., 1990). The antigens of Moore and Lee, as well as the SP-10 immunogen, (described below) were designated "primary vaccine candidates" (Anderson et al., 1987) by the World Health Organization Taskforce on Vaccines for Fertility Regulation.
The sperm-specific antigen, lactic dehydrogenase-C (LDH-C), has been purified, characterized, and used to immunize and inhibit fertility in rabbits (Goldberg, 1973), mice (Lerum and Goldberg, 1974) and baboons (Goldberg et al., 1981). The sperm-specific LDH-C has a substrate specificity different than for muscle and heart LDH and is capable of using branched chain ketoacids as substrates such as a-ketoisovalerate (Blanco et al., 1976) as well as using lactate as substrate. The LDH-C is present in the cytosol and mitochondria of sperm (Montamat et al., 1988) but is also present on the surface of spermatozoa (Erickson et al., 1975) thus providing a basis for the effectiveness of immunization against LDH-C in blocking fertility. More recently, Goldberg and colleagues have cloned the cDNA for the human testes-specific lactate dehydrogenase and characterized its antigenic sites (Millan et al., 1987; Hogrefe et al., 1987; Goldberg, 1987; Hogrefe et al., 1989).
LDH-X, an isozyme of LDH found only in male germ cells, is one of the best characterized human sperm antigens. It has been crystallized and amino acid sequence data is available (Goldberg, 1972). Both auto and iso-immunogenic responses to LDH-X have been noted in mice and rabbits (Goldberg, 1972), although it does not appear to be a potent autoantigen in humans (Goldberg, 1973). Infertility has been seen in baboons inoculated both systemically or locally (intrauterine) with LDH-X (Samuel et al., 1978).
Wright et al. (1989) have identified .lambda.gt11 clones that express the human sperm-specific intra-acrosomal protein antigen SP-10. This antigen is present in the sperm of higher primates and pigs (Herr et al., 1989b). As identified by reactivity with a monoclonal antibody MES-10 (Homyk et al., 1989), the SP-10 antigen has a molecular mass of 28.3 kDa. The SP-10 antigen is not localized to the surface of sperm until after the acrosome reaction but it may be at this point that an antibody interaction with the exposed SP-10 would inhibit sperm-zona pellucida interaction leading to fertilization (Herr et al., 1989b).
An SP-10 fusion protein encoded by 640 nucleotides spanning an immunogenic portion of the SP-10 molecule linked as a fusion protein to a portion of bacterial beta galactosidase has been tested for immunogenicity in rabbits. Rabbits produced polyclonal antibodies which reacted with native SP-10 extracted from human sperm. These antibodies stained the human sperm acrosome. The rabbits did not suffer any ill effects from vaccination (Benjamin, D. C., et al., 1985).
Additionally, production of immunoglobulin A, presumably secretory IgA (sIgA), will block the ability of sperm to penetrate cervical mucus (Kremer and Jager, 1980) as well as inhibit sperm-zona pellucida interactions involved in the fertilization process (Dor et al., 1981; Bronson et al., 1982a, 1982b). A method of immunization that would stimulate a sIgA response, in addition to humoral and cellular immune responses, would therefore be most desirable.
The ovum-specific zona pellucida antigen, ZP-3, is one such antigen that could induce an sIgA response that would result in coating of the zona pellucida with sIgA, thus preventing fertilization by sperm. ZP-3 is unique to the maturing and mature oocytes and is important in sperm binding and induction of the acrosome reaction (Wasserman, 1987). Rabbits, dogs and monkeys immunized with porcine zona pellucida or ZP-3 had abnormal ovarian function and loss of follicles (Wood et al., 1981; Mahi-Brown et al., 1982). However, parenteral immunization of mice (Miller et al., 1989) with a ZP-3 B cell epitope fused to keyhole limpet hemocyanin, induces complete and reversible infertility in Swiss mice, but ovarian autoimmune disease and complete nonreversible infertility of B6AF1 female mice (Tung et al., 1991). Recently it has been possible to separate the epitopes on murine ZP-3 that induce the reversible infertility immune response from the one that induces autoimmune oophoritis (Tung et al., 1991). The use of such a peptide in a vaccine could provide an effective method for blocking fertilization without adverse consequences.
None of the above antigens, however, has been administered to a subject using avirulent carrier microbes. Certain avirulent carrier microbes which include foreign antigens have been shown to induce secretory, humoral and cellular immunities. These strains are developed by the introduction of mutations that cause the bacteria to be substantially incapable of producing functional proteins which are necessary for survival in a host. That is, these avirulent strains do not survive in a manner or for a duration that would cause impairment or a disease state in the host. Such mutants are disclosed in EPO Pub. No. 315,682 (published 17 May 1989), PCT Pub. No. WO 88/09669 (published 15 Dec. 1988) and in Curtiss and Kelly, 1987. Representative are mutants of Salmonella spp. which carry deletion mutations that impair the ability of the bacterium to synthesize adenylate cyclase (ATP pyrophosphate lyase (cyclizing) EC 4.6.1.1) (cya) and the cyclic AMP receptor protein (crp). In addition, removal of the S. typhimurium 91 kb virulence plasmid (Jones et al., 1982) effectively eliminates virulence and lethality following oral inoculation.
Mutants carrying either a point mutation or deletion of the gene encoding beta-aspartic semialdehyde dehydrogenase (asd) have also been developed. This enzyme is found in the mesodiamino-pimelic acid (DAP)-synthesis pathway. DAP is an essential component of peptidoglycan which imparts shape and rigidity to the bacterial cell wall. Bacteria carrying asd mutations can only survive in carefully controlled laboratory environments. Thus, a recombinant vector encoding both asd (an Asd.sup.+ vector) and the antigen of interest, can be placed into an Asd.sup.- carrier cell. Only those cells encoding the desired antigen will survive. The use of such a carrier microbe to deliver a sperm specific antigen could result in an effective method of birth control.